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REVIEW ARTICLE
Year : 2022 | Volume
: 59
| Issue : 1 | Page : 22-28
Morphological and molecular characterization of Aedes aegypti variant collected from Tamil Nadu, India
P Nirmal Kumar1, M Kalimuthu1, M Senthil Kumar1, R Govindrajan1, A Venkatesh1, R Paramasivan1, Ashwani Kumar2, Bhavna Gupta1
1 ICMR-Vector Control Research Centre, Madurai, India
2 ICMR-Vector Control Research Centre, Medical Complex, Puducherry, India
Correspondence Address:
Bhavna Gupta
ICMR-Vector Control Research Centre, 4, Sarojini Street Chinna Chokkikulam, Madurai-625002
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/0972-9062.331413
Background & objectives: Accurate mosquito species identification is the basis of entomological surveys and effective vector control. Mosquito identification is either done morphologically using diagnostic features mentioned in taxonomic keys or by molecular methods using cytochrome oxidase subunit 1 (coxI) and Internal transcribed spacer 2 (ITS2).
Methods: We performed a larval survey for Aedes mosquitoes from eight different geographical regions in Tamil Nadu, India. The mosquitoes collected during the survey were characterized using both morphological and molecular markers.
Results: During an entomological survey from eight different geographical regions in Southern India, a morphological variety named Aedes aegypti var. luciensis was observed. The variant mosquitoes were characterized using both morphological and molecular markers. The variant mosquitoes differed only in the dark scaling of 5th segment of hind-tarsi. Around one third to two third of the 5th segment in variant mosquitoes was dark which has been described as white in identification keys. No other significant difference was observed in adults or immature stages. The variation was heritable and coexisting in the field with the type form mosquitoes. Comparison of the genetic profile of coxI and ITS2 were similar in variant and the type form indicating both of them to be conspecific.
Interpretation & conclusion: The morphological variant mosquitoes were found genetically similar to the Ae. aegypti type form. However, considering its high prevalence and coexistence with Ae. aegypti type form in different geographical regions, detailed studies on bionomics, ecology, genetics, behavior as well as its plausible role in disease transmission are warranted.
Keywords: Aedes aegypti; morphological variant; Aedes aegypti var. luciensis; India; coxI
Morphological and genetic variations within a species (intra-specific) are common in many organisms including mosquitoes. It is generally assumed that the variations having higher adaptive value get selected in certain environmental conditions[1],[2] and due to continuous evolution may lead to speciation. Therefore, these variations if not known properly can affect the species identification which is a basic step in entomological surveys. The careful examination of several mosquito species has identified species complexes which were not known earlier[3],[4]. Species complex is a group of sibling species that are morphologically indistinguishable but they may have different behavioral (preference of host, feeding/biting behavior, resting behavior) and physiological traits (ability to transmit pathogens). For example, there are eight species in Anopheles gambie complex and all the species vary in their behavior and ecology which also impacts their vectorial capacity[5],[6]. Thus, prior information of intra-specific variations is necessary for species identification which forms the basis for accurate interpretation and implementation of the results.
Aedes aegypti which is popularly known as dengue vector is widely spread all over the world. Several named varieties, forms and geographical variations of Ae. aegypti have been documented[7],[8],[9],[10],[11]. However, only two subspecies have been widely recognized namely, Ae. aegypti formosus and Ae. aegypti aegypti[9],[10],[11],[12]. They vary in their ecology, behavior and morphology. Ae. aegypti aegypti is a type form, pale to brownish black, domestic and anthropophilic in nature. Ae. aegypti formosus is a sylvatic ancestral that resides primarily in forest areas and has black appearance of the thorax and abdomen. Moreover, several studies have reported that oral susceptibility of Ae. aegypti to dengue and chikungunya varies with the geographical strains[13],[14]. Thus, the periodic characterization of the mosquito populations and updating the diagnostic features is important for taxonomic purposes as well as to understand the dynamics of the vector borne diseases. The existing variations may affect mosquito biting/feeding traits and the ability to transmit pathogens.
This study was conducted as a part of the nationwide project (SERB India, ECR/2018/001473) to characterize Ae. aegypti populations from different eco-geographical regions in India. In the present study, characterization of Ae. aegypti populations from eight villages in Tamil Nadu, India revealed co-existence of Ae. aegypti type form with the morphologically different variety named as Ae. aegypti var. luciensis.
Material & MethodsSample collection and morphological identification
The present study was carried out in rural and periurban areas in Madurai and nearby districts in Tamil Nadu, India. Geographically, this area extends from N 09°46.568’ to E 078°20.656’ and 078°20.656’ to N’, E 078°17.919 [Table 1]. In this area, total eight sites were investigated. The details of each site are given in [Table 1] and [Figure 1]. Sample collection was done during the months of March–May 2019. All the study sites fall under Tropical Savannah climate and March to July are the hottest months in a year. Each study site was divided into blocks of 1.5 square kilometers that roughly covers continuous stretch of about 40–50 houses and one block was selected randomly for larval survey. Each household in the selected block was inspected for all possible Aedes breeding habitats (e.g., water storage containers, disposables, flower pots, sumps, fridge trays, coolers) and each house was visited only once during the study.
Figure 1: Map of Tamil Nadu, India showing study sites in Madurai district and nearby areas.
Table 1: The details of Aedes mosquito breeding and larval indices at different sites in Tamil Nadu, India.Larval collections from various habitats were done by pipetting method using a Pasteur pipette. The larvae from each breeding habitat were collected in a separate container (~250ml) labeled with house number, habitat type, date of collection and were brought to the laboratory. All the information was entered in the larval collections forms and GPS readings were recorded using handheld GARMIN GPS (etrex 10). The collected larvae were then reared in the laboratory up to adult stage. The adult mosquitoes emerged from each collection were identified under a stereo zoom binocular microscope for generic separation. The species and the sex were identified by following the taxonomic keys[7],[11],[15],[16],[17],[18]. Ae. aegypti mosquitoes were investigated thoroughly to identify any variations in diagnostic features described in the identification keys.
Further to observe the stability and hereditary transmission of the identified morphological variations, Ae. aegypti variant was reared in the laboratory to generate F1. Three different crosses were made to obtain F1 generation, (i) a cross between variant male and variant female, (ii) variant male and type form female and (iii) variant female and type form male. Individual male and female mosquitoes in each cross were kept in mosquito cage. After mating, male mosquitoes were taken out in the test tubes and stored at -20°C. After a period of resting for 2 days, female mosquitoes were fed with chicken blood. After 4–5 days, female mosquitoes laid eggs, which were further reared in the laboratory to generate F1.
Molecular procedures
In order to compliment the morphological identification, total 18 mosquito samples including 11 Ae. aegypti variants, 4 normal Ae. aegypti, 2 Ae. albopictus and 1 Ae. vittatus were characterized using Cytochrome oxidase subunit 1 (coxI). Ae. aegypti variants samples were also characterized using Internal transcribed spacer 2 (ITS2). For this, genomic DNA was extracted from one or two legs of individual mosquito using Blood and Tissue DNA extraction kit (Qiagen, Germany) following manufacturer’s protocol. The DNA concentration was determined using Nanodrop (Thermo Fisher Scientific, USA). DNA was eluted to a final concentration of 10 ng/μl and stored at -20°C. PCR amplification of the coxI gene was performed using the universal forward primer LCO1490 (5’-GGT CAA CAA ATC ATA AGA TAT TGG-3’) and reverse primer HC0219- (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) as described by Folmer et al.[19]. ITS2 region was amplified using primers complementary to the 5.8S and 28S rRNA using primer 5’-ATC ACT CGG CTC ATG GAT CG-3’ and 5’-ATG CTT AAA TTT AGG GGG TAG TC-3’ as described in Weeraratne et al.[20]. PCR amplification was performed by denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 51°C (coxI) and 54°C (ITS2) for 30 sec and elongation at 72°C for 40 sec and the final extension was carried out at 72°C for 7 min. The PCR products were electrophoresed in 2% agarose gel. PCR products showing clear bands of expected size were purified using Wizard SV gel and PCR clean-up kit (Promega Corporation, USA) according to the manufacturer’s protocol. The purified products were then sequenced in both directions using an ABI3730XL automatic sequencer (Europhin Genomics, Bangalore) with the same set of PCR primers.
Three pools from each normal type and the variant type Ae.aegypti mosquitoes were also tested for wolbachia infection using primers WolbF (5′-GAA GAT AAT GAC GGTACT CAC-3′) and Wspecr (5′-AGC TTC GAG TGA AAC CAA TTC-3′) complementary to the wolbachia specific 16s rDNA gene. The gene was amplified following the method described in Carvajal et al. (2019). For wolbachia identification, abdomens from individual adult mosquitoes were separated under microscope and washed with 70% alcohol. Around 10–15 abdomens were pooled together for DNA isolation.
DNA sequence analysis
The DNA sequences of each gene obtained in this study were manually edited using DNASTAR (Lasergene v.10). Comparisons with publicly available sequences were performed using BLAST (www.blast.ncbi.nlm.nih. gov/Blast.cgi) to confirm the species identification. Sequences were aligned by nucleotides and then translated to protein sequences using MEGA X[21]. The number of variable sites, parsimony informative sites, and number of haplotypes were assessed using the DnaSP, version 5.1.10. MEGA version X was used to calculate intra-specific and inter-specific pairwise sequence divergence by Kimura-2 parameter (K2P) distance model[22] also, the evolutionary rates when the divergence times are known. Neighbor-Joining (NJ) phylogenetic trees of coxI and ITS2 sequences were constructed in MEGA X using K2P distances. Reliability of the topology of NJ trees was assessed by bootstrapping using 1000 replicates. Reference sequences for species identification were retrieved from GenBank including, NC 035159.1 for Ae. aegypti, NC 006817.1 for Ae. albopictus and KU380388.1 for Ae. vittatus. All the DNA sequences obtained in this study has been submitted in GenBank database.
Results and DiscussionTotal eight study sites were surveyed to characterize Ae. aegypti populations at both morphological and molecular level. Out of total 296 houses surveyed from eight study sites; 77 houses had positive breeding sources for Aedes mosquitoes [Table 1]. Out of total 778 containers, 110 were found to support the Aedes mosquito breeding [Table 1]. The water storage containers constituted the main breeding habitats for Aedes mosquitoes in our study areas. The major breeding sources observed were plastic drums (48%) followed by plastic containers (27%) and cement tanks (10%) [Figure 2]. The larval indices such as house index and container index varied from 12%–58% and 6.8%–33.3%, respectively, across eight study sites [Table 1]. House and container index provide information on the extent of breeding and intensity of breeding, respectively. House index above 1% is considered critical and an indicator of dengue outbreaks[23],[24]. Both larval indices are high in all the study sites, however, repeated surveys are needed to assess its association with disease outbreaks. Breeding of Aedes mosquitoes especially Ae. aegypti in rural areas is not new. Several reports from different parts of Tamil Nadu have observed high larval indices such as in Tiruchirappalli[25], Tirunelveli[26],[27],[28] and Ramanathpuram districts of Tamil Nadu[29]. This is because of the water storage practices. Due to shortage of water supply, the residents of these areas store water in containers for their needs and these containers constituted the major breeding sources. This is the reason even before rainy season Aedes breeding was observed. The information generated from such entomological surveys indicates that the source reduction could be an effective way to control Aedes population in these areas. The residents should be made aware of these preferred Aedes breeding sites and educated about better water storage practices to avoid Aedes breeding in and around their living areas.
Figure 2: Venn diagram showing percentage of Aedes positive breeding microhabitats/containers observed in all the eight study sites.All the larvae collected from this survey were allowed to grow in laboratory. The morphological characterization of the entire emerged mosquitoes identified only three species of Aedes, namely Ae. aegypti (n=1577), Ae. albopictus (n=8), Ae. vittaus (n=5) [Table 2]. The number of samples obtained area-wise from each species are given in [Table 2]. Ae. aegypti was the predominant species because our survey was restricted in the human dwellings focusing on the man-made containers. In addition to the different species of Aedes genus, we observed a morphological variant of Ae. aegypti. The variant mosquitoes were only different in the aspect of 5th segment hind-tarsi as shown in [Figure 3]. Normally, hind-tarsi 5th segment of Ae. aegypti is completely white in the type form. But, in the variant mosquitoes 1/3rd to 2/3rd of 5th segment of hindtarsi was dark scaled. This variation was observed in both males and females. However, apart from the hind tarsal tip there was no significant variation in other morphological features. Moreover, both the type form and the variant took almost same time to complete their developmental cycle in the laboratory and showed no variation at larval and pupal stages. This variation in Ae. aegypti was first reported by Theobald (1901) in his monograph of the culicidae and by Barraud (1934) in Fauna of British India. Theobald (1901) named it as Ae. aegypti var. luciensis. It has been observed in Demerara. St Lucia, Ancxjilla, Lagos and in India[8],[10],[11],[30]. In Lagos, this variant was observed in rock-holes[30], however, in the present study it was seen coexisting with type form in the same containers. Earlier reports from India observed the variety occasionally[10]. In the present study, this variant was found in all the study sites [Table 2], however, the number of samples varied from 3 to 222 from different sites [Table 2]. It is interesting to note that, this variant was also observed in our previous Ae. aegypti collections from 13 other sites within Madurai corporation area, 4 collections from Port Blair and three cities in Rajasthan (Jaipur, Udaipur and Jodhpur) which remained unnoticed at the time of sample collection and identification. Our data indicates that this variant is well established in India and probably not influenced by the particular geography or any ecological conditions. It is expected that any variation in a population increases if favored by the environment and get selected due to adaptive pressure[2]. Although there is no obvious benefit of the dark scaling on the fifth segment of hind tarsi, this variety might possess other adaptive character (behavioral, ecological or physiological) which needs to be explored. Adaptive feature in a population tend to increase with time, and becomes equally or more prevalent than the other variations.
Figure 3: Aedes aegypti mosquitoes showing variations in 5th segment of hind-tarsi using A. white background B. on black background
Table 2: Species-wise emergence of adult mosquitoes from Aedes larvae collected from eight different study sites in Tamil Nadu, India.Further to observe the stability and the hereditary transmission of the morphological variation, a crossing experiment between normal type and variant mosquitoes was performed. Both the crosses between type form and the variant produced mixed offspring, however, the cross between both variant parents produced only variant mosquitoes in F1 generation. The heritability of this feature is in line with the observations of Summers-Connal[8] where authors observed hereditary transmission of morphological variations in Aedes argenteus, Poiret (synonym of Ae. aegypti). The heritable transmission of this variation indicates that this feature might be genetically controlled and not influenced by ecological or environmental factors. This is the reason this variant is persistent and exists in different regions of India.
In order to compliment the species identification using morphological keys, some of the mosquitoes from each species and the variants were characterized using coxI (699bp length) and ITS2 (393 bp length) genes. coxI is the mitochondrial gene which is used as barcode gene for species identification and ITS2 a phylogenetic marker compliments the coxI gene to identify closely related species. In this study, total 11 Ae. aegypti coxI sequences for variants, 4 from normal Ae. aegypti, 2 from Ae. albopictus and 1 from Ae. vittatus were obtained (GenBank MZ828129-MZ828146). Ae. aegypti sequences matched >99% with GenBank sequences (NC 035159.1) after nucleotide BLAST conforming the species identification. Total 14 SNPs (Single Nucleotide Polymorphism) were identified among 11 variant sequences, of which 11 SNPs were found parsimony informative. Four of these 11 SNPs were non-synonymous in nature. These 11 SNPs were common between variant and normal mosquito sequences. Total 7 haplotypes were identified of which three were shared by both Ae. aegypti types. K2P genetic distance of Ae. aegypti variant with normal type was only 0.0087 while that with Ae. albopictus and Ae. vitattus was 0.13 and 0.11, respectively. The phylogenetic tree constructed from all the 18 coxI sequences along with reference sequences of each species showed clear species-wise separation complying with the morphological identification [Figure 4]A. However, the normal and variant Ae. aegypti clustered together in two clades highlighting their close relationship.
Figure 4: Neighbour-joining phylogenetic tree constructed using A.coxI sequences from Ae. ageypti, var. luciensis, Ae. albopictus and Ae. vittatus B. ITS2 sequences obtained from Ae. aegypti type form and var. luciensis. Bootstrap values > 50 are shown.Similarly, total nine ITS2 sequences were obtained from Ae. aegypti variants (MZ851340-MZ851348). Nucleotide BLAST resulted in >98% similarity with the ITS2 sequences of Ae. aegypti available in GenBank. Total two SNPs were observed and both were parsimony informative. Since ITS2 from Ae. aegypti type form was not sequenced, four ITS2 sequences including KF471587.1 from USA, KF471576.1 from France, KY382418.1 from Sri Lanka and KP2598339.1 from India publicly available in GenBank were retrieved for comparative analysis. The well supported clade contained majority of the var. luciensis sequences clustered together with reference sequences from USA, France and Sri Lanka [Figure 4]B. Significant similarity in the genetic profile of both the genetic markers in normal and variant mosquitoes thus rules out the possibility of genetic isolation between them.
Wolbachia specific 16s gene was sequenced in three pools each from Ae. aegypti type form (42 mosquitoes in 3 pools) and var. luciensis (40 mosquitoes in 3 pools). Only two Ae. aegypti normal type pools amplified band of expected size. However, 16s gene sequences from only one pool could be sequenced successfully (MZ854169). 16s gene of length 920 bp matched significantly (>99%) with publicly available sequences of Wolbachia pipientisstrain (CP041923.1). It was found to be 99.8% similar with the wolbachia strain (MF999263.1) identified recently by Balaji et al.[31] in another city of Tamil Nadu, India. The same strain as an endosymbiont of Ae. aegypti was also reported in USA[32]. The molecular identification of wolbachia in Ae. aegypti has been reported in several studies recently[31],[32],[33],[34]. However, its occurrence in the species is still debatable and only molecular evidence is considered inadequate[34]. The molecular identification is an indicator for further elaborative studies to confirm wolbachia inhabiting Ae. aegypti. This information is particularly important to explore the natural wolbachia infections and to identify the strains circulating in the natural populations before releasing the artificially infected Ae. aegypti mosquitoes in the field as a vector control tool.
In conclusion, the present entomological survey from different geographical regions of India identified a coexisting variety of Ae. aegypti named as Ae. aegypti var. luciensis. Based on crossing experiments and molecular characterization, both the Ae. aegypti type form and the var. luciensis were found to be conspecific. These observations, however, warrants the need for further characterization of variant populations in comparison with the type form to understand its behavior, ecology, genetics and possible role in any pathogen transmission.
AcknowledgementsThe authors would like to thank Dr. P Jambulingam (Former Director, ICMR-VCRC, Puducherry) for valuable suggestions and support in conducting the study. PNK thank Human Resources Development (HRD) Division of ICMR-VCRC, Puducherry. BG acknowledges the funding support provided by Science and Engineering Research Board (SERB) as Early Career Research Award (ECR/2018/001473). All the authors thank Indian Council of Medical Research (ICMR) for intramural support and the facilities. Funding received from Early Career Research Award (ECR/2018/001473) from Science and Engineering Research Board (DST, India).
Conflict of interest: None
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